The goal of this proposal is to use homologous recombination (HR) strategies to elucidate how the chicken beta-globin locus is regulated at the level of chromatin structure, replication timing and origin use, and transcription. Our recent work suggests that the deletion of the mouse and human beta-globin locus control regions (LCRs) does not silence the locus and create the equivalent of a Hispanic thalassemia phenotype in mouse or human. In this proposal, we will perform similar experiments in the chicken beta-globin locus, where locus domain structure, and the presence of a 5' boundary element (insulator) have been well characterized. When sufficient cis regulatory elements have been deleted to created a silenced chicken locus, we will use an addback or knock in approach to determine the minimal sequences involved in activating this loci at the level of transcription, chromatin structure, and DNA replication. These experiments will provide important concepts regarding the function of previously define regulatory elements such as enhancers, LCRs, and insulators as well as identify additional sequences in the chicken beta-globin locus that are important for control of the locus at multiple levels. Inclusion of these sequences in gene therapy vectors may overcome some of the limitations of current viral gene therapy strategies such as low expression levels and loss of expression over time, and thereby aid future efforts towards gene therapy of the hemoglobinopathies and thalassemias. The specific aims of this proposal are: 1. The role of intergenic regulatory sequences in the chicken beta globin locus (beta/epsilon enhancer/LCR) will be investigated using HR mediated deletion in DT40 and subsequent assay in erythroid cells. 2. The role of the upstream hypersensitive sites (HSS) (5'HS1-4) in the chicken beta-globin locus will be investigated including the insulator boundary region (5'HS4). These HSS will be deleted singly and in combination in recombination proficient chicken B cells (DT40) and erythroid cells and their role in the initiation and maintenance of locus chromatin structure and transcription determined. 3. We will use HR mediated deletion in DT40 cells to define essential sequences involved in initiation of DNA replication in the chicken beta- globin locus and the potential role of upstream or other regulatory sequences in DNA replication initiation and timing. 4. Once we have constructed chicken loci silenced for chromatin structure, transcription, and replication in aims 1-4, we will use addback or knockin approaches to reactivate the locus and define minimal and consensus, and unrelated sequences that may have activity. We will also use and addback or knockin approach to determine the ability of chicken regulatory elements to activate silenced mouse and human beta-globin loci.